Hematopoiesis in adult animals is maintained by a small population of clonogenic, multipotent hematopoietic stem cells (HSC), which maintain throughout life the capacity to self-renew and to differentiate to give rise to progeny cells that ultimately generate all lineages of mature blood cells (Kondo et al., Annu Rev Immunol. 2003; 21:759-806. Epub 2002 December 2017). HSC can be purified from both the bone marrow (BM) and blood of mice by fluorescence activated cell sorting (FACS) according to their unique expression of particular cell surface receptors (e.g., c-kit+, Thy1.1loLineage marker−, and Sca-1+ (Morrison and Weissman, Immunity. 1994; 1:661-673; Morrison et al., Development. 1997; 124: 1929-1939), abbreviated KTLS, or KLS and Flk-2− (Christensen and Weissman, Proc Natl Acad Sci USA. 2001; 98:14541-14546. Epub 12001 November 14527), abbreviated KLSF). HSCs can be further divided into long-term hematopoietic stem cells (LTHSC), short-term hematopoietic stem cells (STHSC), and multipotent progenitors (MPP). LTHSC are capable of perpetually repopulating themselves, and also can differentiate into STHSC, which can only repopulate for a finite time period, and non-self renewing MPP.
Upon transplantation, even a single HSC is capable of fully reconstituting hematopoiesis in lethally irradiated recipients, in some cases contributing up to 70% of mature peripheral blood (PB) leukocytes in reconstituted animals (Wagers et al. Science. 2002; 297.2256-2259. Epub 2002 September 2255). This remarkable ability of HSC to functionally regenerate an ablated hematopoietic system forms the basis for bone marrow and PB progenitor cell (PBPC) transplantation, a therapeutic approach that is increasingly employed for the treatment of many diseases, including leukemia and lymphoma (e.g., CML, ALL, AML, and Non-Hodgkin lymphoma), multiple myeloma, breast and ovarian cancers and other solid tumors, bone marrow failure, non-malignant diseases (e.g., aplastic anemia, immune deficiency, and metabolic disorders) and genetic disorders affecting hematopoietic cell function (Weissman, Science. 2000; 287:1442-1446).
In adult mice and humans, the majority of HSC are found in the BM; however, HSC are also constitutively present at low levels in the circulation (see below and Fleming et al., Proc Natl Acad Sci USA. 1993; 90:3760-3764; and Wright et al., Science. 2001; 294:1933-1936). HSCs migrate from the BM to the PB, and likely proliferate in the PB, in a process that is known as “mobilization.” Thus, agents that enhance mobilization can either enhance proliferation in the PB, or enhance migration from the SM to the PB, or both. Mobilization may occur to protect against environmental insult, to reconstitute damaged or depleted hematopoietic system, to maintain a fixed number of HSCs in the bone marrow, and possibly for other reasons as well.
The frequency of HSC in the blood can be significantly increased through the use of “mobilizing” agents, including cytotoxic drugs and/or cytokines, which often act to both drive HSC proliferation and to induce HSC migration from the BM into the bloodstream (Papayannopoulou, Ann NY Acad Sci. 1999; 872:187-197; Wright et al., Blood. 2001; 97:2278-2285). In particular, treatment of mice with a combination of cyclophosphamide (Cy) plus granulocyte-colony stimulating factor (GCSF) induces a rapid and reproducible expansion and migration of HSC (see, e.g., Wright et al., Blood. 2001; 97:2278-2285; Neben et al., Blood. 1993; 81:1960-1967; and Morrison et al., Proc Natl Acad Sci USA. 1997; 94:1908-1913). Following administration of Cy plus 2 daily doses of GCSF, the BM HSC population (referred to as day +2 BM HSC) expands dramatically, reaching about 10-12 times the size of the HSC compartment in normal animals (Morrison et al., 1997; supra).
Expansion of HSC in the early phase of mobilization occurs only in the BM (Wright et al., Blood. 2001; 97:2278-2285), but after day +2, HSC frequency in the BM declines, and HSC begin to appear in significant numbers in the blood and spleen of mobilized animals (Morrison et al., 1997, supra). As noted above, HSC numbers in the blood progressively increase throughout Cy/GCSF-treatment. Previous experiments have suggested that the migration of HSC from the BM to the blood and spleen in the context of Cy/GCSF induced mobilization is tightly coordinated with cell cycle, and that mobilized PB (MPB) HSC of Cy/GCSF-treated mice derive from recently divided BM HSC, which transit through the blood from the BM to the spleen (Wright et al., Blood. 2001; 97:2278-2285). However, both the precise mechanisms by which cell cycle progression is regulated in normal and mobilized HSC and the ways in which proliferation may influence the developmental decisions and migratory capacity of these cells remains unclear.
Bone marrow and PBPC transplantation are increasingly common treatment options for hematopoietic and non-hematopoietic cancers, bone marrow dysfunction, and several other metabolic disorders (Kondo et al., 2003, supra). The success of these transplants critically depends on the surprising ability of intravenously infused HSC to accurately and efficiently home to the BM of transplant recipients and, once there, to expand and differentiate to repopulate the peripheral pool of mature blood cells. As such, hematopoietic reconstitution is a multi-step process, and its efficacy may be limited by the ability of transplanted stem cells to (1) migrate to appropriate BM locations, (2) engraft in available BM niches that support HSC survival and function, and/or (3) self-renew and differentiate to both expand the population of HSC and to regenerate peripheral mature blood cells. At present, mechanisms that control HSC movement between IBM and blood, that regulate the BM microenvironment, or that promote the expansion or differentiation of HSC, remain poorly understood. Additional insights into the factors that regulate these crucial decisions are likely to suggest new strategies to improve the efficacy of this approach and reduce transplant-related mortality.
Furthermore, a substantial overlap has been noted in genes that control the normal function of HSC and those that mediate hematopoietic malignancy, implying in particular that neoplastic progenitor cells may exploit the same or similar mechanisms of proliferation and migration as those normally employed by their non-malignant counterparts (Look, Science. 1997; 278:1059-1064; Lecuyer and Hoang, Experimental Hematology, 2004; 32:11-24.). Thus, an improved understanding of the biology and function of normal HSC may ultimately suggest insights into the ways in which these programs are usurped or dysregulated in oncogenesis and cancer metastasis.